Methods have also been developed to culture pleural mesothelial cells obtained from noncancerous donors. Pure cultures are initiated by pelleting the mesothelial cells from pleural effusion fluid and inoculating the resuspended cells into dishes containing LHC basal nutrient medium supplemented with serum (3%), hydrocortisone (0.5 Mum), insulin (5 Mug/ml) epidermal growth factor (EGF) (5 ng/ml), transferrin (10 Mug/ml), and trace elements. Using this protocol, mesothelial cell cultures have been established from more than 200 donors. The cells can be subcultured at clonal density with a colony-forming efficiency of more than 10% and high density cultures can be subcultured four to six times before senescence. A serum-free medium formulated on the above medium but without serum and in addition containing TGF-beta and PDGF will induce serum/factor-starved cells to undergo one round of DNA synthesis. The cells will continue growing, though at a slow rate, if the latter formula is supplemented with "cell-derived" human IL-1. We have studied the cytopathology of fibers on human mesothelial cells and human fibroblasts in culture. Electron microscopy studies have demonstrated phagocytosis of the fibers and phase-microscopy of metaphase spreads reveal frequent attachment of the fibers to the chromosomes. Investigations regarding the mechanism of asbestos carcinogenesis suggest that oxygen radicals are probably not important intermediates. Asbestos-induced chromosomal abnormalities have also been studied. After one exposure to amosite, a significant increase in chromosomal aberrations was found in mesothelial cells, as compared to unexposed controls. The number of aberrant cells was further increased in cultures treated twice with asbestos, the major aberration type being dicentric chromosomes and acentric fragments.